This invention relates to novel C4xe2x80x3 substituted macrolide derivatives that are useful as antibacterial and antiprotozoa agents in mammals, including man, as well as in fish and birds. This invention also relates to pharmaceutical compositions containing the novel compounds and to methods of treating bacterial and protozoa infections in mammals, fish and birds by administering the novel compounds to mammals, fish and birds requiring such treatment.
Macrolide antibiotics are known to be useful in the treatment of a broad sprectrum of bacterial and protozoa infections in mammals, fish and birds. Such antibiotics include various derivatives of erythromycin A such as azithromycin which is commercially available and is referred to in U.S. Pat. Nos. 4,474,768 and 4,517,359, both of which are incorporated herein by reference in their entirety. Like azithromycin and other macrolide antibiotics, the novel macrolide compounds of the present invention possess potent activity against various bacterial and protozoa infections as described below.
The present invention relates to compounds of the formula 
and to pharmaceutically acceptable salts thereof, wherein:
X is xe2x80x94CH(NR9R10)xe2x80x94, xe2x80x94C(O)xe2x80x94, xe2x80x94C(xe2x95x90NOR9)xe2x80x94, xe2x80x94CH2NR9xe2x80x94, or xe2x80x94N(C1-C6 alkyl)CH2xe2x80x94 wherein the first dash of each of the foregoing X groups is attached to the C-10 carbon of the compound of formula 1 and the last dash of each group is attached to the C-8 carbon of the of the compound of formula 1;
R1 is H, hydroxy or methoxy;
R2 is hydroxy;
R3 is C1-C10 alkyl, C2-C10 alkenyl, C2-C10 alkynyl, cyano, xe2x80x94CH2S(O)nR8 wherein n is an integer ranging from 0 to 2, xe2x80x94CH2OR8, xe2x80x94CH2N(OR9)R8, xe2x80x94CH2NR8R15, xe2x80x94(CH2)m(C6-C10 aryl), or xe2x80x94(CH2)m(5-10 membered heteroaryl), wherein m is an integer ranging from 0 to 4, and wherein the foregoing R3 groups are optionally substituted by 1 to 3 R16 groups;
or R2 and R3 are taken together to form an oxazolyl ring as shown below 
R4 is H, xe2x80x94C(O)R9, xe2x80x94C(O)OR9, xe2x80x94C(O)NR9R10 or a hydroxy protecting group;
R5 is xe2x80x94SR8, xe2x80x94(CH2)nC(O)R8 wherein n is 0 or 1, C1-C10 alkyl, C2-C10 alkenyl, C2-C10 alkynyl, xe2x80x94(CH2)m(C6-C10 aryl), or xe2x80x94(CH2)m(5-10 membered heteroaryl), wherein m is an integer ranging from 0 to 4, and wherein the foregoing R5 groups are optionally substituted by 1 to 3 R16 groups;
each R6 and R7 is independently H, hydroxy, C1-C6 alkoxy, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, xe2x80x94(CH2)m(C6-C10 aryl), or xe2x80x94(CH2)m(5-10 membered heteroaryl), wherein m is an integer ranging from 0 to 4;
each R8 is independently H, C1-C10 alkyl, C2-C10 alkenyl, C2-C10 alkynyl, xe2x80x94(CH2)qCR11R12(CH2)rNR13R14 wherein q and r are each independently an integer ranging from 0 to 3 except q and r are not both 0, xe2x80x94(CH2)m(C6-C10 aryl), or xe2x80x94(CH2)m(5-10 membered heteroaryl), wherein m is an integer ranging from 0 to 4, and wherein the foregoing R6 groups, except H, are optionally substituted by 1 to 3 R16 groups;
or where R8 is as xe2x80x94CH2NR8R15, R15 and R8 may be taken together to form a 4-10 membered saturated monocyclic or polycyclic saturated ring or a 5-10 membered heteroaryl ring, wherein said saturated and heteroaryl rings optionally include 1 or 2 heteroatoms selected from O, S and xe2x80x94N(R8)xe2x80x94, in addition to the nitrogen to which R15 and R8 are attached, said saturated ring optionally includes 1 or 2 carbon-carbon double or triple bonds, and said saturated and heteroaryl rings are optionally substituted by 1 to 3 R16 groups;
each R9 and R10 is independently H or C1-C6 alkyl;
each R11, R12, R13 and R14 is independently selected from H, C1-C10 alkyl, xe2x80x94(CH2)m(C6-C10 aryl), and xe2x80x94(CH2)m(5-10 membered heteroaryl), wherein m is an integer ranging from 0 to 4, and wherein the foregoing R11, R12, R13 and R14 groups, except H, are optionally substituted by 1 to 3 R16 groups;
or R11 and R13 are taken together to form xe2x80x94(CH2)pxe2x80x94 wherein p is an integer ranging from 0 to 3 such that a 4-7 membered saturated ring is formed that optionally includes 1 or 2 carbon-carbon double or triple bonds;
or R13 and R14 are taken together to form a 4-10 membered monocyclic or polycyclic saturated ring or a 5-10 membered heteroaryl ring, wherein said saturated and heteroaryl rings optionally include 1 or 2 heteroatoms selected from O, S and xe2x80x94N(R8)xe2x80x94, in addition to the nitrogen to which R13 and R14 are attached, said saturated ring optionally includes 1 or 2 carbon-carbon double or triple bonds, and said saturated and heteroaryl rings are optionally substituted by 1 to 3 R16 groups;
R15 is H, C1-C10 alkyl, C2-C10 alkenyl, or C2-C10 alkynyl, wherein the foregoing R15 groups are optionally substituted by 1 to 3 substituents independently selected from halo and xe2x80x94OR9;
each R16 is independently selected from halo, cyano, nitro, trifluoromethyl, azido, xe2x80x94C(O)R17, xe2x80x94C(O)OR17, xe2x80x94C(O)OR17, xe2x80x94OC(O)OR17, xe2x80x94NR6C(O)R7, xe2x80x94C(O)NR6R7, xe2x80x94NR6R7, hydroxy, C1-C6 alkyl, C1-C6 alkoxy, xe2x80x94(CH2)m(C6-C10 aryl), and xe2x80x94(CH2)m(5-10 membered heteroaryl), wherein m is an integer ranging from 0 to 4, and wherein said aryl and heteroaryl substituents are optionally substituted by 1 or 2 substituents independently selected from halo, cyano, nitro, trifluoromethyl, azido, xe2x80x94C(O)R17, xe2x80x94C(O)OR17, xe2x80x94C(O)OR17, xe2x80x94OC(O)OR17, xe2x80x94NR6C(O)R7, xe2x80x94C(O)NR6R7, xe2x80x94NR6R7, hydroxy, C1-C6 alkyl, and C1-C6 alkoxy;
each R17 is independently selected from H, C1-C10 alkyl, C2-C10 alkenyl, C2-C10 alkynyl, xe2x80x94(CH2)m(C6-C10 aryl), and xe2x80x94(CH2)m(5-10 membered heteroaryl), wherein m is an integer ranging from 0 to 4;
with the proviso that R8 is not H where R3 is xe2x80x94CH2S(O)nR8.
Preferred compounds of formula 1 include those wherein R1 is hydroxy, R2 is hydroxy, R3 is xe2x80x94CH2NR8R15 or xe2x80x94CH2SR8, and R4 is H.
Other preferred compounds of formula 1 include those wherein R1 is hydroxy, R2 is hydroxy, R3 is xe2x80x94CH2NR8R15, R4 is H, R15 and R8 are each selected from H, C1-C10 alkyl, C2-C10 alkenyl, and C2-C10 alkynyl, wherein said R15 and R8 groups, except H, are optionally substituted by 1 or 2 substituents independently selected from hydroxy, halo and C1-C6 alkoxy. Specific preferred compounds having the foregoing general structure include those wherein R15 is either H or is selected from the following groups from which R8 is also independently selected: methyl, ethyl, allyl, n-butyl, isobutyl, 2-methoxyethyl, cyclopentyl, 3-methoxypropyl, 3-ethoxypropyl, n-propyl, isopropyl, 2-hydroxyethyl, cyclopropyl, 2,2,2-trifluoroethyl, 2-propynyl, sec-butyl, tert-butyl, and n-hexyl.
Other preferred compounds of formula 1 include those wherein R1 is hydroxy, R2 is hydroxy, R3 is xe2x80x94CH2NHR8, R4 is H, and R8 is xe2x80x94(CH2)m(C6-C10 aryl) wherein m is an integer ranging from 0 to 4. Specific preferred compounds having the foregoing general structure include those wherein R8 is phenyl or benzyl.
Other preferred compounds of formula 1 include those wherein R1 is hydroxy, R2 is hydroxy, R3 is xe2x80x94CH2NR15R8, R4 is H, and R15 and R8 are taken together to form a saturated ring. Specific preferred compounds having the foregoing general structure include those wherein R15 and R8 are taken together to form a piperidino, trimethyleneimino, or morpholino ring.
Other preferred compounds of formula 1 include those wherein R1 is hydroxy, R2 is hydroxy, R3 is xe2x80x94CH2NR15R8, R4 is H, and R15 and R8 are taken together to form a heteroaryl ring optionally substituted by 1 or 2 C1-C6 alkyl groups. Specific preferred compounds having the foregoing general structure include those wherein R15 and R8 are taken together to form a pyrrolidino, triazolyl, or imidazolyl ring wherein said heteroaryl groups are optionally substituted by 1 or 2 methyl groups.
Other preferred compounds of formula 1 include those wherein R1 is hydroxy, R2 is hydroxy, R3 is xe2x80x94CH2SR8, R4 is H, and R8 is selected from C1-C10 alkyl, C2-C10 alkenyl, and C2-C10 alkynyl, wherein said R8 groups are optionally substituted by 1 or 2 substituents independently selected from hydroxy, halo and C1-C6 alkoxy. Specific preferred compounds having the foregoing general structure include those wherein R8 is methyl, ethyl, or 2-hydroxyethyl.
Other preferred compounds of formula 1 include those wherein R1 is hydroxy, R2 is hydroxy, R4 is H, and R3 is selected from C1-C10 alkyl, C2-C10 alkenyl, and C2-C10 alkynyl, wherein said R3 groups are optionally substituted by 1 or 2 substituents independently selected from hydroxy, xe2x80x94C(O)R17, xe2x80x94NR6R7, halo, cyano, azido, 5-10 membered heteroaryl, and C1-C6 alkoxy. Specific preferred compounds having the foregoing general structure include those wherein R3 is methyl, allyl, vinyl, ethynyl, 1-methyl-1-propenyl, 3-methoxy-1-propynyl, 3-dimethylamino-1-propynyl, 2-pyridylethynyl, 1-propynyl, 3-hydroxy-1-propynyl, 3-hydroxy-1-propenyl, 3-hydroxypropyl, 3-methoxy-1-propenyl, 3-methoxypropyl, 1-propynyl, n-butyl, ethyl, propyl, 2-hydroxyethyl, formylmethyl, 6cyano-1-pentynyl, 3-dimethylamino1-propenyl, or 3-dimethylaminopropyl.
Other preferred compounds of formula 1 include those wherein R1 is hydroxy, R2 is hydroxy, R4 is H, and R3 is xe2x80x94(CH2)m(5-10 membered heteroaryl) wherein m is an integer ranging from 0 to 4. Specific preferred compounds having the foregoing general structure include those wherein R3 is 2-thienyl, 2-pyridyl, 1-methyl-2-imidazolyl, 2-furyl, or 1-methyl-2-pyrrolyl.
Other preferred compounds of formula 1 include those wherein R1 is hydroxy, R2 is hydroxy, R4 is H, and R3 is xe2x80x94(CH2)m(C6-C10 aryl) wherein m is an integer ranging from 0 to 4. Specific preferred compounds having the foregoing general structure include those wherein R3 is phenyl.
Specific compounds of formula 1 include those wherein R2 and R3 are taken together to form an oxazolyl ring as shown below 
wherein R5 is as defined above.
Specific compounds of formula 1 include those wherein R3 is selected from the following: 
wherein X3 is O, S or xe2x80x94N(R15)xe2x80x94, and wherein the xe2x80x94OR9 group may be attached at any available carbon on the phenyl group.
The invention also relates to a pharmaceutical composition for the treatment of a bacterial infection or a protozoa infection in a mammal, fish, or bird which comprises a therapeutically effective amount of a compound of formula 1, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.
The invention also relates to a method of treating a bacterial infection or a protozoa infection in a mammal, fish, or bird which comprises administering to said mammal, fish or bird a therapeutically effective amount of a compound of formula 1 or a pharmaceutically acceptable salt thereof.
The term xe2x80x9ctreatmentxe2x80x9d, as used herein, unless otherwise indicated, includes the treatment or prevention of a bacterial infection or protozoa infection as provided in the method of the present invention.
As used herein, unless otherwise indicated, the terms xe2x80x9cbacterial infection(s)xe2x80x9d and xe2x80x9cprotozoa infection(s)xe2x80x9d include bacterial infections and protozoa infections that occur in mammals, fish and birds as well as disorders related to bacterial infections and protozoa infections that may be treated or prevented by administering antibiotics such as the compounds of the present invention. Such bacterial infections and protozoa infections, and disorders related to such infections, include the following: pneumonia, otitis media, sinusitus, bronchitis, tonsillitis, and mastoiditis related to infection by Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, Staphylococcus aureus, or Peptostreptococcus spp.; pharynigitis, rheumatic fever, and glomerulonephritis related to infection by Streptococcus pyogenes, Groups C and G streptococci, Clostridium diptheriae, or Actinobacillus haemolyticum; respiratory tract infections related to infection by Mycoplasma pneumoniae, Legionella pneumophila, Streptococcus pneumoniae, Haemophilus influenzae, or Chlamydia pneumoniae; uncomplicated skin and soft tissue infections, abscesses and osteomyelitis, and puerperal fever related to infection by Staphylococcus aureus, coagulase-positive staphylococci (i.e., S. epidermidis, S. hemolyticus, etc.), Streptococcus pyogenes, Streptococcus agalactiae, Streptococcal groups C-F (minute-colony streptococci), viridans streptococci, Corynebacterium minutissimum, Clostridium spp., or Bartonella henselae; uncomplicated acute urinary tract infections related to infection by Staphylococcus saprophyticus or Enterococcus spp.; urethritis and cervicitis; and sexually transmitted diseases related to infection by Chlamydia trachomatis, Haemophilus ducreyi, Treponema pallidum, Ureaplasma urealyticum, or Neiserria gonorrheae; toxin diseases related to infection by S. aureus (food poisoning and Toxic shock syndrome), or Groups A, B, and C streptococci; ulcers related to infection by Helicobacter pylori; systemic febrile syndromes related to infection by Borrelia recurrentis; Lyme disease related to infection by Borrelia burgdorferi; conjunctivitis, keratitis, and dacrocystitis related to infection by Chlamydia trachomatis, Neisseria gonorrhoeae, S. aureus, S. pneumoniae, S. pyogenes, H. influenzae, or Listeria spp.; disseminated Mycobacterium avium complex (MAC) disease related to infection by Mycobacterium avium, or Mycobacterium intracellulare; gastroenteritis related to infection by Campylobacter jejuni, intestinal protozoa related to infection by Cryptosporidium spp.; odontogenic infection related to infection by viridans streptococci; persistent cough related to infection by Bordetella pertussis; gas gangrene related to infection by Clostridium perfringens or Bacteroides spp.; and atherosclerosis related to infection by Helicobacter pylori or Chlamydia pneumoniae. Bacterial infections and protozoa infections and disorders related to such infections that may be treated or prevented in animals include the following: bovine respiratory disease related to infection by P. haem., P. multocida, Mycoplasma bovis, or Bordetella spp.; cow enteric disease related to infection by E. coli or protozoa (i.e., coccidia, cryptosporidia, etc.); dairy cow mastitis related to infection by Staph. aureus, Strep. uberis, Strep. agalactiae, Strep. dysgalactiae, Klebsiella spp., Corynebacterium, or Enterococcus spp.; swine respiratory disease related to infection by A. pleuro., P. multocida, or Mycoplasma spp.; swine enteric disease related to infection by E. coli, Lawsonia intracellularis, Salmonella, or Serpulina hyodyisinteriae; cow footrot related to infection by Fusobacterium spp.; cow metritis related to infection by E. coli, cow hairy warts related to infection by Fusobacterium necrophorum or Bacteroides nodosus; cow pink-eye related to infection by Moraxella bovis; cow premature abortion related to infection by protozoa (i.e. neosporium); urinary tract infection in dogs and cats related to infection by E. coli; skin and soft tissue infections in dogs and cats related to infection by Staph. epidermidis, Staph. intermedius, coagulase neg. Staph. or P. multocida; and dental or mouth infections in dogs and cats related to infection by Alcaligenes spp., Bacteroides spp., Clostridium spp., Enterobacter spp., Eubacterium, Peptostreptococcus, Porphyromonas, or Prevotella. Other bacterial infections and protozoa infections and disorders related to such infections that may be treated or prevented in accord with the method of the present invention are referred to in J. P. Sanford et al., xe2x80x9cThe Sanford Guide To Antimicrobial Therapy,xe2x80x9d 26th Edition, (Antimicrobial Therapy, Inc., 1996).
The present invention also relates to a method of preparing the above compound of formula 1, or a pharmaceutically acceptable salt thereof, wherein R3 is xe2x80x94CH2S(O)nR8, xe2x80x94CH2OR8 or xe2x80x94CH2NR8R15, wherein n, R15 and R8 are as defined above with the proviso that R8 is not H where R3 is xe2x80x94CH2S(O)nR8, which comprises treating a compound of the formula 
wherein X, R1 and R4 are as defined above, with a compound of the formula HSR8, HOR8 or HNR15R8, wherein n, R15 and R8 are as defined above, optionally followed by oxidation of the xe2x80x94SR8 substituent to form xe2x80x94S(O)R8 or xe2x80x94S(O)2R8.
In a further aspect of the above process of preparing the compound of formula 1, or a pharmaceutically acceptable salt thereof, the above compound of formula 3 is prepared by treating a compound of the formula 
wherein X, R1 and R4 are as defined above, with (CH3)3S(O)nX2, wherein n is 0 or 1 and X2 is halo, xe2x80x94BF4 or xe2x80x94PF6, preferably iodo or xe2x80x94BF4, in the presence of a base such as as potassium tert-butoxide, sodium tert-butoxide, sodium ethoxide, sodium hydride, 1,1,3,3-tetramethylguanidine, 1,8-diazabicyclo[5.4.0]undec-7-ene, 1,5-diazabicylo[4.3.0]non-5-ene, potassium hexamethyidisilazide (KHMDS), potassium ethoxide, or sodium methoxide, preferably KHMDS or a sodium-containing base such as sodium hydride.
The present invention also relates to the above compounds of formulas 2 and 3 which, as indicated above, are useful in the preparation of the above compounds of formula 1 and pharmaceutically acceptable salts thereof.
The term xe2x80x9chydroxy protecting groupxe2x80x9d, as used herein, unless otherwise indicated, includes acetyl, benzyloxycarbonyl, and various hydroxy protecting groups familiar to those skilled in the art including the groups referred to in T. W. Greene, P. G. M. Wuts, xe2x80x9cProtective Groups In Organic Synthesis,xe2x80x9d (J. Wiley and Sons, 1991).
The term xe2x80x9chaloxe2x80x9d, as used herein, unless otherwise indicated, includes fluoro, chloro, bromo or iodo.
The term xe2x80x9calkylxe2x80x9d, as used herein, unless otherwise indicated, includes saturated monovalent hydrocarbon radicals having straight, cyclic or branched moieties, or mixtures thereof. It is to be understood that where cyclic moieties are intended, at least three carbons in said alkyl must be present. Such cyclic moieties include cyclopropyl, cyclobutyl and cyclopentyl.
The term xe2x80x9calkoxyxe2x80x9d, as used herein, unless otherwise indicated, includes xe2x80x94O-alkyl groups wherein alkyl is as defined above.
The term xe2x80x9carylxe2x80x9d, as used herein, unless otherwise indicated, includes an organic radical derived from an aromatic hydrocarbon by removal of one hydrogen, such as phenyl or naphthyl.
The term xe2x80x9c5-10 membered heteroarylxe2x80x9d, as used herein, unless otherwise indicated, includes aromatic heterocyclic groups containing one or more heteroatoms each selected from O, S and N, wherein each heterocyclic group has from 5 to 10 atoms in its ring system. Examples of suitable 5-10 membered heteroaryl groups include pyridinyl, imidazolyl, pyrimidinyl, pyrazolyl, (1,2,3,)- and (1,2,4)-triazolyl, pyrazinyl, tetrazolyl, furyl, thienyl, isoxazolyl, oxazolyl, pyrrolyl and thiazolyl.
The phrase xe2x80x9cpharmaceutically acceptable salt(s)xe2x80x9d, as used herein, unless otherwise indicated, includes salts of acidic or basic groups which may be present in the compounds of the present invention. The compounds of the present invention that are basic in nature are capable of forming a wide variety of salts with various inorganic and organic acids. The acids that may be used to prepare pharmaceutically acceptable acid addition salts of such basic compounds of are those that form non-toxic acid addition salts, i.e., salts containing pharmacologically acceptable anions, such as the hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, isonicotinate, acetate, lactate, salicylate, citrate, acid citrate, tartrate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate and pamoate [i.e., 1,1xe2x80x2-methylene-bis-(2-hydroxy-3naphthoate)] salts. The compounds of the present invention that include an amino moiety may form pharmaceutically acceptable salts with various amino acids, in addition to the acids mentioned above.
Those compounds of the present invention that are acidic in nature are capable of forming base salts with various pharmacologically acceptable cations. Examples of such salts include the alkali metal or alkaline earth metal salts and, particularly, the calcium, magnesium, sodium and potassium salts of the compounds of the present invention.
Certain compounds of the present invention may have asymmetric centers and therefore exist in different enantiomeric and diastereomic forms. This invention relates to the use of all optical isomers and stereoisomers of the compounds of the present invention, and mixtures thereof, and to all pharmaceutical compositions and methods of treatment that may employ or contain them.
The present invention includes the compounds of the present invention, and the pharmaceutically acceptable salts thereof, wherein one or more hydrogen, carbon or other atoms are replaced by isotopes thereof. Such compounds may be useful as research and diagnostic tools in metabolism pharmacokinetic studies and in binding assays.
The compounds of of the present invention may be prepared according to Schemes 1-3 below and the description that follows. In the following Schemes, unless otherwise indicated, substituents X, R1, R2, R3 , R4, R5, R6, R7, R8, R9, R10, R11, R12, R13, R14, R15, R16 and R17 are as defined above. 
This invention uses a variety of macrolide templates as starting materials. They include azithromycin, erythromycin, clarithromycin, erythromycylamine as well as their analogs. Azithromycin can be prepared according to methods described in U.S. Pat. Nos. 4,474,768 and 4,517,359, referred to above. Erythromycin can be prepared, or isolated, according to methods described in U.S. Pat. Nos. 2,653,899 and 2,823,203. Clarithromycin can be prepared according to methods described in U.S. Pat. No. 4,331,803.
The foregoing starting materials require proper functional group protection before various modifications can take place, and deprotection after desired modifications are complete. The most commonly used protecting groups for amino moieties in the macrolide compounds of this invention are benzyloxycarbonyl (Cbz) and t-butyloxycarbonyl (Boc) groups. Hydroxyl groups are generally protected as acetates or Cbz carbonates. The relative reactivity of various hydroxyl groups in the macrolide molecules of the general type claimed in this invention has been well established. Such differences in reactivity permit selective modification of different parts of the compounds of this invention.
In above Schemes, the C-2xe2x80x2 hydroxy group (R4 is H) is selectively protected by treating the macrolide compound with one equivalent of acetic anhydride in dichloromethane in the absence of external base to provide the corresponding compound wherein R4 is acetyl. The acetyl protecting group may be removed by treating the compound of formula 3 with methanol at 23-65xc2x0 C. for 10-48 hours. The C-2xe2x80x2 hydroxy may also be protected with other protecting groups familiar to those skilled in the art, such as the Cbz group. Where X is xe2x80x94CH2NHxe2x80x94, the C-9 amino group may also require protection before further synthetic modifications are performed. Suitable protecting groups for the amino moiety are Cbz and Boc groups. To protect the C-9 amino group, the macrolide may be treated with t-butyl dicarbonate in anhydrous tetrahydrofuran (THF) or benzyloxycarbonyl N-hydroxysuceinimide ester or benzylchloroformate to protect the amino group as its t-butyl or benzyl carbamate. Both the C-9 amino and C-2xe2x80x2 hydroxy may be selectively protected with the Cbz group in one step by treating the compound of formula 2 with benzylchloroformate in THF and water. The Boc group may be removed by acid treatment and the Cbz group may be removed by conventional catalytic hydrogenation. In the following description, it is assumed that, where X is xe2x80x94CH2NHxe2x80x94, the C-9 amino moiety as well as the C-2xe2x80x2 hydroxy group are protected and deprotected as would be deemed appropriate by those skilled in the art.
In Scheme 1, the compound of formula 2 may be prepared according to methods familiar to those skilled in the art, including one or more methods described in the Journal of Antibiotics, 1988, pages 1029-1047. In step 1 of Scheme 1, the compound of formula 2 is treated with R3MgX1 or R3xe2x80x94Li and Mg(X1)2, wherein X1 is a halide such as chloro or bromo, in a solvent such as THF, ethylene glycol dimethyl ether (DME), diisopropyl ether, toluene, diethyl ether, or tetramethylethylenediamine (TMEDA), hexanes, or a mixture of two or more of the foregoing solvents, preferably an ether solvent, at a temperature ranging from about xe2x88x9278xc2x0 C. to about room temperature (20-25xc2x0 C.), to provide the compound of formula 1 wherein R2 is hydroxy and R1, R3 and R4 are as defined above.
Scheme 2 illustrates the preparation of compounds of formula 1 through use of an epoxide intermediate. In step 1 of Scheme 2, the compound of formula 3 may be generated by two methods. In one method (Method A), the compound of formula 2 is treated with (CH3)3S(O)X2, wherein X2 is halo, xe2x80x94BF4 or xe2x80x94PF6, preferably iodo, in the presence of a base such as as potassium tert-butoxide, sodium ethoxide, sodium tert-butoxide, sodium hydride, 1,1,3,3-tetramethylguanidine, 1,8-diazabicyclo[5.4.0]undec-7-ene, 1,5-diazabicylo[4.3.0]non-5-ene, potassium ethoxide, or sodium methoxide, preferably a sodium-containing base such as sodium hydride, in a solvent such as THF, an ether solvent, dimethylformamide (DMF), or methyl sulfoxide (DMSO), or a mixture of two or more of the foregoing solvents, at a temperature within the range of about 0xc2x0 C. to about 60xc2x0 C., to provide the compound of formula 3 in which the following configuration of the epoxide moiety predominates 
In a second method (Method B), the compound of formula 2 is treated with (CH3)3SX2, wherein X2 is halo, xe2x80x94BF4 or xe2x80x94PF6, preferably xe2x80x94BF4, in the presence of a base such as as potassium tert-butoxide, sodium tert-butoxide, sodium ethoxide, sodium hydride, 1,1,3,3-tetramethylguanidine, 1,8-diazabicyclo[5.4.0]undec-7-ene, 1,5-diazabicylo[4.3.0]non-5ene, potassium ethoxide, potassium hexamethyldisilazide (KHMDS) or sodium methoxide, preferably KHMDS, in a solvent such as THF, an ether solvent, DMF, or DMSO, or a mixture of two or more of the foregoing solvents, at a temperature within the range of about 0xc2x0 C. to about 60xc2x0 C., to provide the compound of formula 3 in which the following configuration of the epoxide moiety predominates 
In step 2 of Scheme 2, the compound of formula 3 may be converted to a compound of formula 1 wherein R2 is hydroxy and R3 is a group that is attached to the C-4xe2x80x3 carbon through a methylene group, such as where R3 is xe2x80x94CH2NR15R8 or xe2x80x94CH2S(O)nR8 wherein n, R15 and R8 are as defined above. To prepare a compound of formula 1 wherein R3 is xe2x80x94CH2NR15R8, the compound of formula 3 may be treated with a compound of the formula HNR15R8, wherein R15 and R8 are as defined above, in the absence or presence of a polar solvent such as water, methanol, or THF, or a mixture of the foregoing solvents, at a temperature ranging from about room temperature to about 100xc2x0 C., preferably about 60xc2x0 C., optionally in the presence of a halide reagent such as potassium iodide, lithium perchlorate, magnesium perchlorate, lithium tetrafluoroborate, pyridinium hydrochloride, or a tetraalkylammonium halide reagent such as tetrabutylammonium iodide. To prepare a compound of formula 1 wherein R3 is xe2x80x94CH2S(O)nR8 wherein n and R8 are as defined above, the compound of formula 3 may be treated with a compound of the formula HSR8 in the presence of K2CO3, Kl, or sodium methoxide, in an aromatic solvent such as methanol, benzene or toluene at a temperature ranging from about room temperature to about 120xc2x0 C. As appropriate, the sulfur moiety may be oxidized to xe2x80x94SOxe2x80x94 or SO2xe2x80x94 according to methods familiar to those skilled in the art. To prepare a compound of formula 1 wherein R3 is xe2x80x94CH2SR8 and R8 is xe2x80x94(CH2)qCR11R12(CH2)rNR13R14, wherein the substituents of said R8 group are as defined above, the compound of formula 3 may be treated with a compound of the formula HSxe2x80x94(CH2)qCR11R12(CH2)rxe2x80x94NPhth, wherein NPhth represents phthalimido, and potassium iodide to provide the compound of formula 1 wherein R3 is xe2x80x94CH2S(CH2)qCR11R12(CH2)rNH2, after removal of the phthalimido moiety, which may be further modified as necessary. By an analogous method, a compound of formula 1 wherein R3 is xe2x80x94CH2NR15R8 and R8 is xe2x80x94(CH2)qCR11R12(CH2)rNR13R14 may be prepared by treating the compound of formula 3 with either a compound of the formula HNR9xe2x80x94(CH2)qCR11R12(CH2)rxe2x80x94NR13R14 or a compound of the formula H2Nxe2x80x94(CH2)qCR11R12(CH2)rxe2x80x94NH2 followed by reductive alkylation of the nitrogen atoms. Using the same or an analogous method, a compound of formula 1 wherein R3 is xe2x80x94CH2OR8 and R8 is as defined above may be prepared by treating a compound of formula 3 with a compound of the formula HOR8.
Scheme 3 illustrates the preparation of compounds of formula 1 in which R2 and R3 are taken together to form an oxazolyl moiety. In step 1 of Scheme 3, the compound of formula 3 is treated with sodium azide in the presence of NH4Cl in methanol or water, or a mixture of the two solvents, at a temperature ranging from about 0xc2x0 C. to about 100xc2x0 C., preferably about 80xc2x0 C., to provide the compound of formula 4. In step 2 of Scheme 3, the compound of formula 4 may be converted to the corresponding amine of formula 5 via conventional catalytic hydrogenation. Preferably, such hydrogenation is done using Pd (10% on carbon) powder under an H2 atmosphere (1 atm). The resulting amine of formula 5 may be converted to various compounds of formula 1 wherein R3 is xe2x80x94CH2NR15R8 using conventional synthetic methods such as reductive amination.
In step 3 of Scheme 3, the compound of formula 5 may be converted to the compound of formula 1 wherein R2 and R3 are taken together as shown by treating the compound of formula 5 with a compound of formula R5xe2x80x94CN, R5xe2x80x94Cxe2x95x90N(OCH3), R5xe2x80x94Cxe2x95x90N(OC2H5), R5xe2x80x94C(O)Cl, or R5xe2x80x94CO2H, wherein R5 is as defined above, except it is not NH2, in the presence or absence of an acid, such as Hcl, or a Lewis acid, such as ZnCl2 or BF4Et3O, or a base, such as NaOH or TEA, in a solvent such as THF, a chlorohydrocarbon (such as CH2Cl2 or chlorobenzene), at a temperature ranging from about room temperature to reflux. To prepare the corresponding compound where R5 is amino, the compound of formula 5 is treated with BrCN and sodium acetate in methanol at a temperature ranging from about room temperature to reflux. In the alternative, the compound of formula 5 may proceed as indicated in steps 4 and 5 of Scheme 3. In step 4 of Scheme 3, the compound of formula 5 is treated with thiocarbonyldiimidazole in methylene chloride at a temperature ranging from about 0xc2x0 C. to room temperature to provide the compound of formula 25. In step 5 of Scheme 3, the compound of formula 25 is treated with R5xe2x80x94X1, wherein X1 is a halide such as bromo or iodo, and a base such as sodium methoxide in a solvent such as methanol or acetone at a temperature ranging from about 0xc2x0 C. to room temperature.
The compounds of the present invention may have asymmetric carbon atoms and therefore exist in different enantiomeric and diastereomeric forms. Diastereomeric mixtures can be separated into their individual diastereomers on the basis of their physical chemical differences by methods known to those skilled in the art, for example, by chromatography or fractional crystallization. Enantiomers may be separated by converting the enantiomeric mixtures into a diastereomeric mixture by reaction with an appropriate optically active compound (e.g., alcohol), separating the diastereomers and converting (e.g., hydrolyzing) the individual diastereomers to the corresponding pure enantiomers. Such separations may also be accomplished through use of standard chiral HPLC. The use of all such isomers, including diastereomer mixtures and pure enantiomers, are considered to be part of the present invention.
The compounds of the present invention that are basic in nature are capable of forming a wide variety of different salts with various inorganic and organic acids. Although such salts must be pharmaceutically acceptable for administration to mammals, it is often desirable in practice to initially isolate the compound of the present invention from the reaction mixture as a pharmaceutically unacceptable salt and then simply convert the latter back to the free base compound by treatment with an alkaline reagent and subsequently convert the latter free base to a pharmaceutically acceptable acid addition salt. The acid addition salts of the base compounds of this invention are readily prepared by treating the base compound with a substantially equivalent amount of the chosen mineral or organic acid in an aqueous solvent medium or in a suitable organic solvent, such as methanol or ethanol. Upon careful evaporation of the solvent, the desired solid salt is readily obtained. The desired salt can also be precipitated from a solution of the free base in an organic solvent by adding to the solution an appropriate mineral or organic acid.
Those compounds of the present invention that are acidic in nature are capable of forming base salts with various cations. For compounds that are to be administered to mammals, fish or birds such salts must be pharmaceutically acceptable. Where a pharmaceutically acceptable salt is required, it may be desirable to initially isolate the compound of the present invention from the reaction mixture as a pharmaceutically unacceptable salt and then simply convert the latter to a pharmaceutically acceptable salt in a process analogous to that described above relating to the conversion of pharmaceutically unacceptable acid addition salts to pharmaceutically acceptable salts. Examples of base salts include the alkali metal or alkaline-earth metal salts and particularly the sodium, amine and potassium salts. These salts are all prepared by conventional techniques. The chemical bases which are used as reagents to prepare the pharmaceutically acceptable base salts of this invention are those which form non-toxic base salts with the acidic compounds of the present invention. Such non-toxic base salts include those derived from such pharmacologically acceptable cations as sodium, potassium, calcium, magnesium, various amine cations, etc. These salts can easily be prepared by treating the corresponding acidic compounds with an aqueous solution containing the desired pharmacologically acceptable bases with cations such as sodium, potassium, calcium, magnesium, various amine cations, etc., and then evaporating the resulting solution to dryness, preferably under reduced pressure. Alternatively, they may also be prepared by mixing lower alkanolic solutions of the acidic compounds and the desired alkali metal alkoxide together, and then evaporating the resulting solution to dryness in the same manner as before. In either case, stoichiometric quantities of reagents are preferably employed in order to ensure completeness of reaction and maximum yields of the desired final product.
The antibacterial and antiprotozoa activity of the compounds of the present invention against bacterial and protozoa pathogens is demonstrated by the compound""s ability to inhibit growth of defined strains of human (Assay I) or animal (Assays II and III) pathogens.
Assay I, described below, employs conventional methodology and interpretation criteria and is designed to provide direction for chemical modifications that may lead to compounds that circumvent defined mechanisms of macrolide resistance. In Assay I, a panel of bacterial strains is assembled to include a variety of target pathogenic species, including representatives of macrolide resistance mechanisms that have been characterized. Use of this panel enables the chemical structure/activity relationship to be determined with respect to potency, spectrum of activity, and structural elements or modifications that may be necessary to obviate resistance mechanisms. Bacterial pathogens that comprise the screening panel are shown in the table below. In many cases, both the macrolide-susceptible parent strain and the macrolide-resistant strain derived from it are available to provide a more accurate assessment of the compound""s ability to circumvent the resistance mechanism. Strains that contain the gene with the designation of ermA/ermB/ermC are resistant to macrolides, lincosamides, and streptogramin B antibiotics due to modifications (methylation) of 23S rRNA molecules by an Erm methylase, thereby generally prevent the binding of all three structural classes. Two types of macrolide efflux have been described; msrA encodes a component of an efflux system in staphylococci that prevents the entry of macrolides and streptogramins while mefA/E encodes a transmembrane protein that appears to efflux only macrolides. Inactivation of macrolide antibiotics can occur and can be mediated by either a phosphorylation of the 2xe2x80x2-hydroxyl (mph) or by cleavage of the macrocyclic lactone (esterase). The strains may be characterized using conventional polymerase chain reaction (PCR) technology and/or by sequencing the resistance determinant The use of PCR technology in this application is described in J. Sutcliffe et al., xe2x80x9cDetection Of Erythromycin-Resistant Determinants By PCRxe2x80x9d, Antimicrobial Agents and Chemotherapy, 40(11), 2562-2566 (1996). The assay is performed in microtiter trays and interpreted according to Performance Standards for Antimicrobial Disk Susceptibility Testsxe2x80x94Sixth Edition; Approved Standard, published by The National Committee for Clinical Laboratory Standards (NCCLS) guidelines; the minimum inhibitory concentration (MIC) is used to compare strains. Compounds are initially dissolved in dimethylsulfoxide (DMSO) as 40 mg/ml stock solutions.
Assay II is utilized to test for activity against Pasteurella multocida and Assay III is utilized to test for activity against Pasteurella haemolytica. 
This assay is based on the liquid dilution method in microliter format. A single colony of P. multocida (strain 59A067) is inoculated into 5 ml of brain heart infusion (BHI) broth. The test compounds are prepared by solubilizing 1 mg of the compound in 125 xcexcl of dimethylsulfoxide (DMSO). Dilutions of the test compound are prepared using uninoculated BHI broth. The concentrations of the test compound used range from 200 xcexcg/ml to 0.098 xcexcg/ml by two-fold serial dilutions. The P. multocida inoculated BHI is diluted with uninoculated BHI broth to make a 104 cell suspension per 200 xcexcl. The BHI cell suspensions are mixed with respective serial dilutions of the test compound, and incubated at 37xc2x0 C. for 18 hours. The minimum inhibitory concentration (MIC) is equal to the concentration of the compound exhibiting 100% inhibition of growth of P. multocida as determined by comparison with an uninoculated control.
This assay is based on the agar dilution method using a Steers Replicator. Two to five colonies isolated from an agar plate are inoculated into BHI broth and incubated overnight at 37xc2x0 C. with shaking (200 rpm). The next morning, 300 xcexcl of the fully grown P. haemolytica preculture is inoculated into 3 ml of fresh BHI broth and is incubated at 37xc2x0 C. with shaking (200 rpm). The appropriate amounts of the test compounds are dissolved in ethanol and a series of two-fold serial dilutions are prepared. Two ml of the respective serial dilution is mixed with 18 ml of molten BHI agar and solidified. When the inoculated P. haemolytica culture reaches 0.5 McFarland standard density, about 5 xcexcl of the P. haemolytica culture is inoculated onto BHI agar plates containing the various concentrations of the test compound using a Steers Replicator and incubated for 18 hours at 37xc2x0 C. Initial concentrations of the test compound range from 100-200 xcexcg/ml. The MIC is equal to the concentration of the test compound exhibiting 100% inhibition of growth of P. haemolytica as determined by comparison with an uninoculated control.
The in vivo activity of the compounds of formula (I) can be determined by conventional animal protection studies well known to those skilled in the art, usually carried out in mice.
Mice are allotted to cages (10 per cage) upon their arrival, and allowed to acclimate for a minimum of 48 hours before being used. Animals are inoculated with 0.5 ml of a 3xc3x97103 CFU/ml bacterial suspension (P. Multocida strain 59A006) intraperitoneally. Each experiment has at least 3 non-medicated control groups including one infected with 0.1xc3x97challenge dose and two infected with 1xc3x97challenge dose; a 10xc3x97challenge data group may also be used. Generally, all mice in a given study can be challenged within 30-90 minutes, especially if a repeating syringe (such as a Cornwall(copyright) syringe) is used to administer the challenge. Thirty minutes after challenging has begun, the first compound treatment is given. It may be necessary for a second person to begin compound dosing if all of the animals have not been challenged at the end of 30 minutes. The routes of administration are subcutaneous or oral doses. Subcutaneous doses are administered into the loose skin in the back of the neck whereas oral doses are given by means of a feeding needle. In both cases, a volume of 0.2 ml is used per mouse. Compounds are administered 30 minutes, 4 hours, and 24 hours after challenge. A control compound of known efficacy administered by the same route is included in each test. Animals are observed daily, and the number of survivors in each group is recorded. The P. multocida model monitoring continues for 96 hours (four days) post challenge.
The PD50 is a calculated dose at which the compound tested protects 50% of a group of mice from mortality due to the bacterial infection which would be lethal in the absence of drug treatment.
The compounds of formula 1, and the pharmaceutically acceptable salts thereof (hereinafter xe2x80x9cthe active compoundsxe2x80x9d), may be adminstered through oral, parenteral, topical, or rectal routes in the treatment of bacterial and protozoa infections. In general, these compounds are most desirably administered in dosages ranging from about 0.2 mg per kg body weight per day (mg/kg/day) to about 200 mg/kg/day in single or divided doses (i.e., from 1 to 4 doses per day), although variations will necessarily occur depending upon the species, weight and condition of the subject being treated and the particular route of administration chosen. However, a dosage level that is in the range of about 4 mg/kg/day to about 50 mg/kg/day is most desirably employed. Variations may nevertheless occur depending upon the species of mammal, fish or bird being treated and its individual response to said medicament, as well as on the type of pharmaceutical formulation chosen and the time period and interval at which such administration is carried out. In some instances, dosage levels below the lower limit of the aforesaid range may be more than adequate, while in other cases still larger doses may be employed without causing any harmful side effects, provided that such larger doses are first divided into several small doses for administration throughout the day.
The active compounds may be administered alone or in combination with pharmaceutically acceptable carriers or diluents by the routes previously indicated, and such administration may be carried out in single or multiple doses. More particularly, the active compounds may be administered in a wide variety of different dosage forms, i.e., they may be combined with various pharmaceutically acceptable inert carriers in the form of tablets, capsules, lozenges, troches, hard candies, powders, sprays, creams, salves, suppositories, jellies, gels, pastes, lotions, ointments, aqueous suspensions, injectable solutions, elixirs, syrups, and the like. Such carriers include solid diluents or fillers, sterile aqueous media and various non-toxic organic solvents, etc. Moreover, oral pharmaceutical compositions can be suitably sweetened and/or flavored. In general, the active compounds are present in such dosage forms at concentration levels ranging from about 5.0% to about 70% by weight.
For oral administration, tablets containing various excipients such as microcrystalline cellulose, sodium citrate, calcium carbonate, dicalcium phosphate and glycine may be employed along with various disintegrants such as starch (and preferably corn, potato or tapioca starch), alginic acid and certain complex silicates, together with granulation binders like polyvinylpyrrolidone, sucrose, gelatin and acacia. Additionally, lubricating agents such as magnesium stearate, sodium lauryl sulfate and talc are often very useful for tabletting purposes. Solid compositions of a similar type may also be employed as fillers in gelatin capsules; preferred materials in this connection also include lactose or milk sugar as well as high molecular weight polyethylene glycols. When aqueous suspensions and/or elixirs are desired for oral adinistration, the active compound may be combined with various sweetening or flavoring agents, coloring matter or dyes, and, if so desired, emulsifying and/or suspending agents as well, together with such diluents as water, ethanol, propylene glycol, glycerin and various like combinations thereof.
For parenteral administration, solutions of an active compound in either sesame or peanut oil or in aqueous propylene glycol may be employed. The aqueous solutions should be suitably buffered (preferably pH greater than 8) if necessary and the liquid diluent first rendered isotonic. These aqueous solutions are suitable for intravenous injection purposes. The oily solutions are suitable for intraarticular, intramuscular and subcutaneous injection purposes. The preparation of all these solutions under sterile conditions is readily accomplished by standard pharmaceutical techniques will known to those skilled in the art.
Additionally, it is also possible to administer the active compounds of the present invention topically and this may be done by way of creams, jellies, gels, pastes, patches, ointments and the like, in accordance with standard pharmaceutical practice.
For administration to animals other than humans, such as cattle or domestic animals, the active compounds may be administered in the feed of the animals or orally as a drench composition.
The active compounds may also be adminstered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles and multilamellar vesicles. Liposomes can be formed from a variety of phospholipids, such as cholesterol, stearylamine or phosphatidylcholines.
The active compounds may also be coupled with soluble polymers as targetable drug carriers. Such polymers can include polyvinylpyrrolidone, pyran copolymer, polyhydroxypropylmethacrylamide phenyl, polyhydroxyethylaspartamide-phenol, or polyethyleneoxide-polylysine substituted with palmitoylresidues. Furthermore, the active compounds may be coupled to a class of biodegradable polymers useful in achieving controlled release of a drug, for example, polylactic acid, polyglycolic acid, copolymers of polylactic and polyglycolic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates and cross-linked or amphipathic block copolymers of hydrogels.